CHAPTER OUTLINE

The general haematology laboratory performs numerical, morphological and biochemical analysis of blood samples (now often as part of a combined blood sciences laboratory). Blood samples must be anticoagulated, for example with ethylenediamine tetra-acetic acid (EDTA) for determination of full blood count and reticulocyte count, blood film examination and plasma viscosity and tri-sodium citrate for coagulation tests and measurement of erythrocyte sedimentation rate.

The full blood count (FBC) is the most requested test profile in haematology. It comprises:

• red blood cell (RBC) count

• red cell indices (volume and haemoglobin content)

• white blood cell (WBC) count

• white blood cell differential count (neutrophils, lymphocytes, monocytes, eosinophils and basophils)

• platelet count.

Full blood counts are usually performed on fully automated analysers, which provide accurate quantitation of cell parameters and can generate alerts, commonly known as ‘flags’, to the presence of unusual characteristics that may warrant review of a blood film.

Blood cells are counted and sized either by electrical impedance or laser light scattering methods. The impedance method utilizes the fact that blood cells are very poor conductors of electricity. When a cell passes through a small aperture across which an electric current is applied, there is a measurable rise in the electrical impedance, with the pulse height being proportional to the volume of the cell. Each cell generates a separate impulse. Light scatter utilizes the fact that cells scatter light when passed through a laser beam: the number of cells can be counted and information about the cells can be obtained according to the pattern of scatter. White cell counting is performed after lysis of the red cells.

Clumping of cells results in underestimation of cell numbers. This may occur, for example, as a result of autoagglutination of red cells in cold agglutinin disease or in vitro clumping of platelets because of inadequate mixing with EDTA anticoagulant.

The red cell indices are a combination of measured and derived red cell parameters. The red cell indices usually reported are:

• haematocrit (HCT) or packed cell volume (PCV), the percentage volume of red cells in the blood

• mean cell haemoglobin (MCH), the average mass of haemoglobin per red cell

• mean cell haemoglobin concentration (MCHC), the average concentration of haemoglobin per red cell.

Some analysers measure the MCV and calculate the HCT or PCV; others measure the HCT and calculate the MCV.

is useful when assessing anaemia. For example, it is reduced in iron deficiency anaemia and increased in megaloblastic anaemia.

is reduced in iron deficiency anaemia and thalassaemias; further tests such as ferritin and HbA₂ measurement may be performed to differentiate between these two conditions.

is reduced in iron deficiency but is probably a more useful tool within the laboratory than clinically. It is very stable in health, so it can be used as an internal quality control; a significant change in the daily mean MCHC indicates a drift in analyser calibration or a fault in its function. The MCHC is also useful for detecting anomalous results: a falsely raised Hb concentration, for example as a consequence of lipaemia, or a falsely reduced RBC count, for example owing to red cell autoagglutination, will result in a falsely raised MCHC.

The formulae for the derived parameters are as follows:

The main function of platelets is to form a plug at sites of damage to vascular endothelium. A low platelet count (thrombocytopenia) may result from decreased production, increased consumption or abnormal pooling in the spleen. Decreased production is a consequence of either general marrow failure or selective depression, for example by viral infection, drugs and chemicals. Platelet consumption is a feature of certain types of autoimmune diseases and occurs in disseminated intravascular coagulation (which is often a result of septicaemia) and thrombotic thrombocytopenic purpura. A high platelet count (thrombocytosis) may be seen as part of a reactive process such as infection or in myeloproliferative neoplasms. Automated haematology analysers routinely measure the platelet count by impedance or laser light scattering; the reference method uses flow cytometry after labelling the platelets with CD41 or CD61 antibodies.

Reticulocytes are immature red cells, which are released from the bone marrow into the circulation one to two days before maturation. An increase in the reticulocyte count is indicative of an increase in red cell production (erythropoiesis) to meet physiological demand, for example in haemolytic anaemia.

Reticulocytes contain ribosomal RNA, which can be detected by two main methods. When unfixed red cells are incubated with dyes such as new methylene blue, the ribosomal RNA precipitates out and appears as a blue reticular network within the cells, which can be visualised using a light microscope, allowing the reticulocytes to be counted. Most automated FBC analysers now perform reticulocyte counts by flow cytometry, using a fluorescent dye that binds to the ribosomal RNA; the number of reticulocytes can be expressed as a percentage of total red cells and as an absolute count.

expressed in mm/h, is a measure of the rate at which red blood cells settle when an anticoagulated blood sample is left to stand in a column.

is usually measured by an automated viscometer. The plasma is drawn through a capillary tube under constant pressure and temperature; the rate of flow is measured and expressed as viscosity of plasma relative to distilled water.

Measurement of ESR or plasma viscosity may be useful when assessing the acute phase response. Both are affected by the presence of large proteins such as fibrinogen, α₂-macroglobulin and immunoglobulins, and are therefore usually raised during an acute phase response. They are non-specific screening tools for inflammation, and are useful in monitoring of diseases such as rheumatoid arthritis and response to treatment for example with non-steroidal anti-inflammatory drugs and anti-tumour necrosis factor.

Plasma viscosity has several advantages over ESR. For example, it is not affected by physiological and environmental factors. Measurement is rapid, reproducible and lends itself better to quality assurance procedures than measurement of ESR. Conversely, ESR is a cheap test, but is influenced by factors such as anaemia, sex, age and (in females) stage of the menstrual cycle and must be carried out within about six hours of venepuncture.

White cells express characteristic nuclear, cytoplasmic and surface antigens; this is referred to as the immunophenotype of the cell. Immunophenotyping has been made possible by the availability of a large number of antibodies, which target specific cellular antigens. For example, antibodies that are directed against the CD19 antigen will bind to B lymphocytes.

Infectious mononucleosis (IM), also known as glandular fever, is caused by the Epstein–Barr virus (EBV), which infects B lymphocytes. It is associated with a high titre of a heterophile antibody, so-called because it also reacts with red cells of other species, for example sheep, ox and horse. This antibody has been termed the Paul–Bunnell antibody, named after John Paul and Walls Bunnell who discovered them.

The Paul–Bunnell antibody is not adsorbed by guinea pig kidney, in contrast to other heterophile antibodies (Forssman antibodies). Differential adsorption tests utilize this principle; the patient’s serum is incubated with ox red cells and separately with guinea pig kidney antigen, after which horse red cells (reagent) are added to the mixtures. Red cell agglutination only in the latter mixture indicates the presence of the Paul–Bunnell antibody.

An alternative test is the latex agglutination test. A reagent containing latex microspheres coated with purified Paul–Bunnell antigen is mixed with the patient’s serum; agglutination indicates presence of the Paul–Bunnell antibody.

Definitive confirmation of EBV infection requires serological detection of a rise in the titre of specific anti-EBV capsid antigen IgM, which occurs in the first few weeks following infection.

Microscopic examination of a well stained blood film is an essential part of many haematological investigations. The film may be made either manually or by an automated slide maker. After being air dried, it is stained with Romanowsky dyes such as May–Grünwald Giemsa or modified Wright stain, which contain azure B and eosin. This enables the assessment of the size, shape, maturity and contents of blood cells by light microscopy. Blood film examination is useful in the investigation of the causes of anaemia and to diagnose white cell disorders such as leukaemia and parasitic infections such as malaria. Less commonly, a blood film may be requested for specific reasons such as to look for acanthocytes (red cells with irregular spicules), which are associated with abetalipoproteinaemia, liver disease and several rare degenerative neurological diseases.